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Related Experiment Video

Updated: May 14, 2026

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis
08:22

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and λ) Hyperspectral FRET Imaging and Analysis

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Quantifying spillover spreading for comparing instrument performance and aiding in multicolor panel design.

Richard Nguyen1, Stephen Perfetto, Yolanda D Mahnke

  • 1Flow Cytometry Core, Vaccine Research Center, NIAID, NIH, Bethesda, Maryland 20892-3015, USA.

Cytometry. Part a : the Journal of the International Society for Analytical Cytology
|February 8, 2013
PubMed
Summary

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Spillover spreading (SS) in flow cytometry causes measurement errors. A new metric, the spillover spreading matrix (SSM), quantifies these errors, improving instrument quality control and multicolor panel development.

Area of Science:

  • Flow cytometry
  • Immunofluorescence
  • Analytical chemistry

Background:

  • Measurement errors in flow cytometry, termed spillover spreading (SS), arise from fluorescence signal bleed-through between detectors.
  • SS can negatively impact assay sensitivity and is influenced by instrument configuration, reagents, and signal intensity.

Purpose of the Study:

  • To develop a novel, intensity-independent metric to quantify spillover spreading between detectors.
  • To establish the spillover spreading matrix (SSM) as a tool for assessing instrument performance and predicting multicolor panel behavior.

Main Methods:

  • Devised a metric to quantify SS between detector pairs, independent of fluorescence intensity.
  • Combined pairwise SS values into a spillover spreading matrix (SSM) for each instrument.

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Last Updated: May 14, 2026

Measurement of 3-Dimensional cAMP Distributions in Living Cells using 4-Dimensional (x, y, z, and &lambda;) Hyperspectral FRET Imaging and Analysis
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Published on: October 27, 2020

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Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

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  • Determined SSM using single-stained controls across multiple instruments over time and varying signal quality.

    • Main Results:

    • The SSM quantifies fluorescence spectrum interactions that limit reagent sensitivity.
    • SSM values are highly reproducible (CV < 30% over 2 months) and comparable between similar instruments.
    • Instrument-specific SSM differences highlight underperforming detectors.

    Conclusions:

    • The SSM is a reliable metric for quantifying spillover spreading in flow cytometry.
    • Monitoring the SSM is crucial for instrument quality control, ensuring consistent sensitivity and performance.
    • The SSM aids in optimizing and developing multicolor immunofluorescence panels by predicting their performance.